Project Summary/Abstract TAZ/YAP are transcriptional effectors and proto-oncoproteins of the Hippo pathway, a signal transduction pathway regulating tumor growth and metastasis in multiple cancers including sarcomas. While much has been uncovered with regards to the upstream regulation of TAZ and YAP, little is known about epigenetic regulation of their transcriptional programs, representing a significant gap in knowledge. To address this gap, we utilized the TAZ-CAMTA1 (TC) and YAP-TFE3 (YT) fusion proteins. TC and YT are driving oncoproteins in epithelioid hemangioendothelioma (EHE), a sarcoma arising in adolescents (responsive to PA-16-251, Gene Fusions in Pediatric Sarcomas). TC and YT, hyperactivated forms of TAZ and YAP, are relevant because gene fusions are the most common type of genetic alterations of TAZ/YAP in cancer. Our objective in this proposal is to determine if TC and YT further stimulate TAZ/YAP transcription by altering the epigenome. Our central hypothesis is that TC and YT recruit YEATS2 and ZZZ3, part of the Ada2a-containing (ATAC) histone acetyltransferase complex, which potentiate the TAZ/YAP oncogenic transcriptional programs. This hypothesis is supported by two serial unbiased approaches: BioID mass spectrometry followed by an shRNA screen that identified YEATS2 and ZZZ3 as the proteins most critical for TC driven anchorage independent growth. We plan to test our central hypothesis with the following specific aims utilizing TC, the predominant fusion protein in EHE (85% of EHE): Aim 1) Determine how YEATS2 and ZZZ3 alter the TAZ-CAMTA1 transcriptional program. We will perform ChIP-seq and determine if TC co-localizes with YEATS2 and ZZZ3 on the genome. RNA-seq will be performed in TC expressing cell lines with and without expression of YEATS2 or ZZZ3 to identify their effects on the TC transcriptome. Aim 2) Identify domains of TAZ-CAMTA1, YEATS2, and ZZZ3 required for cellular transformation. The interaction of YEATS2 and ZZZ3 with TC will be dissected by co-immunoprecipitation utilizing deletion mutants. The mutants will be further studied in anchorage independent growth assays we have developed. Aim 3) Determine the contributions of the YEATS2 and ZZZ3 interactions with TAZ-CAMTA1 in vivo. We will use both genetic and pharmacological approaches to inhibit YEATS2/ZZZ3/ATAC function in novel TC transgenic and xenograft sarcoma mouse models and evaluate their effect on tumorigenesis and metastasis. The proposal is innovative because it utilizes TC and YT as well as two serial unbiased approaches to identify YEATS2/ZZZ3/ATAC as novel epigenetic regulators of the TAZ/YAP transcriptome, expanding the focus of the field and addressing a significant gap in knowledge. The project is significant because it identifies YEATS2 and ZZZ3 as novel oncoproteins and drivers in sarcoma and provides a rationale for inhibiting histone modifying complexes as a novel way of targeting the Hippo pathway, which currently lacks a therapeutic target.